Transient aggregation of ubiquitinated proteins is a cytosolic unfolded protein response to inflammation and endoplasmic reticulum stress

Abstract

Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-κB and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress.

FIGURE 1.

Inflammation and ER stress up-regulate p62 and induce ALIS formation. BMMs were incubated with LPS (100 ng/ml) for 2 h (A) or 8 h (B and C), infected with BCG for 1 h and then kept in culture for 1 h (A) or 7 h (B and C), or treated with thapsigargin (TG, 5 μm) or tunicamycin (TM, 5 μg/ml) for 12 h (A–C). Total RNA was analyzed by Real-Time PCR using primers specific for p62 (A). Cell lysates were analyzed by immunoblot using antibodies against p62 or β-actin (B) or examined by immunofluorescence microscopy using ubiquitin antibody to demonstrate ALIS (green) or p62 antibody (red) (C). Quantitation of the percentage of cells expressing ALIS are indicated. Data represent mean ± S.D., n = 3 (A) or 5 (B and C). **, p < 0.001, compared with control cells. Scale bar, 5 μm.

FIGURE 2.

p62 is required for ALIS formation. BMMs obtained from wild type or p62 knock-out mice incubated for 8–12 h with LPS (100 ng/ml), thapsigargin (TG, 5 μm), tunicamycin (TM, 5 μg/ml) or puromycin (8 μg/ml), or were infected with BCG for 1 h followed by culture in regular medium for 7 h. Cells lysates were analyzed by immunoblot using antibodies against p62 or β-actin (A), or examined by immunofluorescence microscopy using ubiquitin antibody to demonstrate ALIS or p62 antibody (red) (B). Quantitations of the percentage of cells expressing ALIS are indicated. Data represent mean ± S.D., n = 5. **, p < 0.001, compared with control cells. Scale bar, 5 μm.

FIGURE 3.

NF-κB signaling pathway is required for ALIS formation. BMMs obtained from wild type or p50 knock-out mice were incubated for 8–12 h (A) or 1–8 h (B and C) with LPS (100 ng/ml), thapsigargin (TG, 5 μm), tunicamycin (TM, 5 μg/ml), or puromycin (8 μg/ml), or were infected with BCG for 1 h followed by culture in regular medium for 7 h. Cells were examined by immunofluorescence microscopy using ubiquitin antibody to demonstrate ALIS (green) or p62 antibody (red) (A). Quantitation of the percentage of cells expressing ALIS is indicated. Total RNA was analyzed by Real-Time PCR using primers specific for p62 (B). Cell lysates were subjected to immunoblot analysis using indicated antibodies (C). Data represent mean ± S.D., n = 5 (A) or 3 (C). **, p < 0.001. Scale bar, 5 μm.

FIGURE 4.

XBP1 knockdown enhances ALIS formation. RAW246.7 cells stably expressing control shRNA, or XBP1 shRNA were untreated (Control) or treated with thapsigargin (TG, 5 μm) for 12 h. Total RNA was analyzed by Real-Time PCR using primers specific for XBP1 (A), sec61α (C), and p62 (D), or RT-PCR using primers specific for XBP1 (B). E, cells lysates were analyzed by immunoblot using antibodies against p62 or β-actin. F, quantification of p62 protein levels from experiments of E. G, cells were examined by immunofluorescence microscopy using ubiquitin antibody to demonstrate ALIS (green) or p62 antibody (red). Quantitation of the percentage of cells expressing ALIS is indicated. Data represent mean ± S.D., n = 3–5. *, p < 0.05.; **, p < 0.001. Scale bar, 5 μm.

FIGURE 5.

ALIS formation and clearance are independent of ATG7. BMMs from wild type (ATG7^F/F) or ATG7 knock-out (ATG7^F/FCre⁺) mice were infected with BCG for 1 h followed by culture in regular medium for 7 h (BCG8h) or 35 h (BCG36h). In some experiments, BMMs were infected with BCG for 1 h followed by culture in regular medium for 7 h and then treated for 4 h with 25 μm cycloheximide either with or without 200 nm of bafilomycin A. Cell lysates were subjected to immunoblot analysis using antibodies against ATG7, p62, LC3B, or β-actin (A). Cells were examined by immunofluorescence microscopy using ubiquitin antibody to demonstrate ALIS (green) or p62 antibody (B). Quantitation of the percentage of cells expressing ALIS is shown (C). Data represent mean ± S.D., n = 5. **, p < 0.001. Scale bar, 5 μm.

FIGURE 6.

ALIS are independent of LC3B, Beclin 1, or Rab9. A and B, BMMs from wild type or LC3B knock-out mice were infected with BCG for 1 h followed by culture in regular medium for 7 h (BCG8h) or 48 h (BCG48h). Cell lysates were subjected to immunoblot analysis using antibodies against LC3B, p62, or β-actin (A). Quantitation of the percentage of cells expressing ALIS by immunofluorescence microscopy (B). C and D, RAW264.7 cells were transfected for 48 h with control siRNA, Beclin1 siRNA, or Rab9 siRNA. Cells were incubated for 12 h with thapsigargin (TG, 5 μm) or infected with BCG for 1 h followed by culture in regular medium for 7 h. Cell lysates were subjected to immunoblot analysis using antibodies against Beclin1, Rab9, p62, or β-actin (C). Quantitation of the percentage of cells expressing ALIS by immunofluorescence microscopy using ubiquitin (D). Data represent mean ± S.D., n = 5. **, p < 0.001, compared with control cells.

FIGURE 7.

Working model: p62 and NF-κB mediates ALIS in response to inflammation and ER stress. TLR activation initiated by pathogen infection and/or inflammation and ER stress initiated by accumulation of unfolded proteins activate mTOR signaling to increased protein synthesis, which is unavoidably associated with production of unfolded proteins in the cytosol. Inflammation is also associated with ER stress and UPR in the ER serves to reduce protein translation and increase protein refolding and degradation. ER stress is also associated with “leakage” of unfolded proteins to the cytosol. p62 recruits unfolded proteins in the cytosol to form ALIS as an adaptive mechanism to altered proteostasis. There is a reciprocal regulation between p62 and NF-κB. p62 is important for sustained NF-κB activation, and NF-κB is required for p62 mRNA transcription induced by ER stress or inflammation.